A two-photon scanning confocal microscope is requested for integration with a patch clamp setup. This integrated system will be used for experiments that combine imaging with simultaneous electrophysiological recording from living cells. Six investigators, Ed Chapman, Roberto Coronado, Robert Fettiplace, Meyer Jackson, Jeff Walker, and Lea Ziskind-Conhaim form a core of major users of this system. All of these investigators have expertise in both electrophysiology and imaging, and all have similar needs for a system that can combine laser scanning microscopy and patch clamping. They will investigate various aspects of signaling in cardiac myocytes, hair cells, presynaptic terminals, and neuroendocrine terminals. Calcium plays vital roles in the physiology of each of these systems. Most of the experiments to be conducted primarily with calcium imaging to obtain high resolution information about calcium signals in real time as cells are subject to electrical stimulation. Each of the six investigators has NIH funding for research that can be dramatically enhanced by this instrument. Among other advantages, this system will enable Chapman to investigate the regulation of fusion pores by synaptotagmin, Coronado to investigate Ca 2+dynamics at individual Ca 2+release sites, Fettiplace to study Ca 2+ in individual stereocilia, Jackson to investigate spatial variations in Ca 2+dynamics in nerve terminals, Walker to apply caged signaling molecules to T-tubules and Ziskind-Conhaim to investigate the role of specific cell types in spinal rhythmicity. The shared instrument grant will enhance all of these research programs by providing better signal-to-noise in Ca 2+ imaging, better tissue penetration in imaging experiments in slices, and better localization in caged compound photolysis.